Treating various disorders using trkb agonists

ABSTRACT

Novel compounds and methods for activating the TrkB receptor are provided. The methods include administering in vivo or in vitro a therapeutically effective amount of a compound containing four six-membered rings and a substituted or unsubstituted C 5  or C 6  heteroaryl or heterocycloalkyl ring and pharmaceutically acceptable salts, prodrugs, and derivatives thereof. Specifically, methods and compounds for the treatment of disorders including neurologic, neuropsychiatric, and metabolic disorders are provided. For example, a method is provided of treating or reducing the risk of depression, anxiety, or obesity in a subject, which includes selecting a subject with or at risk of developing depression, anxiety, or obesity, and administering to the subject a therapeutically effective amount of the described compounds. A further method of promoting neuroprotection in a subject is provided, which includes selecting a subject in need of neuroprotection, and administering to the subject a therapeutically effective amount of the described compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application and claims the benefit ofU.S. patent application Ser. Nos. 13/479,653 filed May 24, 2012 and13/056,377 filed Jan. 28, 2011, which are a national stage applicationof PCT/US2009/051966 filed Jul. 28, 2009, and claims the benefit of U.S.Provisional Application Ser. No. 61/084,117, filed Jul. 28, 2008, and61/118,907, filed Dec. 1, 2008. The entire disclosures of the priorapplications are hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grant No.RO1-NS045627 from the National Institutes of Health. The government hascertain rights in this invention.

BACKGROUND

Neurologic and neuropsychiatric disorders such as depression, anxiety,amyotrophic lateral sclerosis, and central nervous system injuries, toname a few, afflict millions of people every year resulting in amultitude of symptoms including weight change, decreased energy,headaches, digestive problems, chronic pain, paralysis, and in certaininstances, death.

One class of growth factors proposed as a treatment for neurologic andneuropsychiatric disorders are neurotrophins, which includebrain-derived neurotrophic factor (BDNF). BDNF is believed to haveneurotrophic action on various neuronal populations including sensoryneurons, motor neurons, dopaminergic neurons of the substantia nigra,and cholinergic neurons of the basal forebrain, which are involved inseveral neurologic and neuropsychiatric disorders. Preclinical evidenceindicates that BDNF might be useful as a therapeutic agent for variousneurologic and neuropsychiatric disorders; however, the in vivoinstability of such a peptide based therapy limits its usefulness.

Neurotrophins are also indicated in metabolic disorders. Mutations inthe tyrosine kinase receptor trkB or in one of its natural ligands,e.g., BDNF or neurotrophin-4 (NT4), are known to lead to severehyperphagia and obesity in rodents and humans. Administration of trkBligands such as BDNF or NT4 have been shown to suppress appetite andbody weight in a dose-dependent manner in several murine models ofobesity. Accumulating evidence indicates that TrkB signaling directlymodulates appetite, metabolism, and taste preference. TrkB agonists thusemerge as potential therapeutics for metabolic disorders.

SUMMARY

Novel compounds and methods for the treatment of disorders includingneurologic disorders, neuropsychiactric disorders (e.g., anxiety ordepression), and metabolic disorders (e.g., obesity) are provided. Themethods include administering to a subject a therapeutically effectiveamount of a compound having the following formula:

or a pharmaceutically acceptable salt or prodrug thereof. In thiscompound, R¹ and R² are each independently selected from hydrogen,substituted or unsubstituted C₁₋₁₂ alkyl, substituted or unsubstitutedC₁₋₁₂ haloalkyl, substituted or unsubstituted C₂₋₁₂ alkenyl, substitutedor unsubstituted C₂₋₁₂ alkynyl, substituted or unsubstituted aryl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, substituted or unsubstituted cycloalkylalkyl, andsubstituted or unsubstituted heterocycloalkylalkyl; R³ is hydrogen,carbonyl, hydroxyl, —O—R¹, —O—C(═O)—R¹, or —NR⁵R⁶, wherein R⁵ and R⁶,are each independently selected from R¹; R⁴ is carbonyl, —R¹, —O—R¹, or—O—C(═O)—R¹; A is a substituted or unsubstituted C₅ or C₆

heteroaryl or C₅ or C₆ heterocycloalkyl;

is a single or double bond, wherein two double bonds are not adjacent;and

is a double bond or

A first method for the treatment of disorders including neurologicdisorders, neuropsychiactric disorders, and metabolic disorders usingthis compound is related to treating or reducing the risk of depression,anxiety, or obesity in a subject, which includes selecting a subjectwith or at risk of developing depression, anxiety, or obesity, andadministering to the subject a therapeutically effective amount of thecompound described above or a derivative thereof. A further method ofpromoting neuroprotection in a subject is provided, which includesselecting a subject in need of neuroprotection, and administering to thesubject a therapeutically effective amount of the compound describedabove or a derivate thereof.

A method of activating a TrkB receptor on a neuron also is provided. Themethod includes providing the neuron with a TrkB receptor, thencontacting the TrkB receptor in vitro with the compound described aboveor a derivate thereof in an amount sufficient to activate the TrkBreceptor. The neuron can be, for example, a mammalian cell.

The details of one or more examples of the compounds and methods are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages will be apparent from the descriptionand drawing, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows a design for a chemical screen to identify TrkB agonists.

FIG. 2 shows the chemical structures of 12 gedunin related compounds.

FIG. 3 shows the results of an apoptosis inhibitory assay for the 12gedunin related compounds from FIG. 2.

FIG. 4 shows the results of an apoptosis inhibitory assay underoxygen-glucose-deprivation conditions (left panel) and a titration assayfor deoxygedunin under the same conditions for the 12 gedunin relatedcompounds from FIG. 2.

FIG. 5 shows immunofluorescent staining results illustrating neuronalTrkB phosphorylation by various compounds.

FIG. 6 shows Western blots illustrating TrkB phosphorylation usingvarious compounds.

FIG. 7 shows Western blots illustrating activation time (left panels)and dose dependency (right panels) for deoxygedunin activation of Erk1/2and Akt.

FIG. 8 shows Western blots illustrating deoxygedunin activation of theTrkB receptor.

FIG. 9 shows Western blots illustrating TrkB phosphorylation bydeoxygedunin over time.

FIG. 10 shows TrkB oral activation in mouse brain by both8-dihydroxyflavone and deoxygedunin.

FIG. 11 shows immunofluorescent staining results illustrating TrkBactivation by deoxygedunin in the hippocampus.

FIGS. 12A (top panel) and 12B (bottom panel) show graphs illustratingbinding activity for [³H]-deoxygedunin to various domains of TrkB.

FIG. 13 shows Scatchard plot analysis of deoxygedunin provocation ofTrkB dimerization.

FIG. 14 shows Western blots illustrating the results of a GST pull-downassay for TrkB and various compounds including deoxygedunin.

FIG. 15 shows Western blots of a TrkB truncation assay for deoxygeduninbinding.

FIG. 16 shows Western blots illustrating that deoxygedunin elicitedtyrosine phosphorylation in TrkB but not TrkA or TrkC receptors intransfected HEK293 cells.

FIG. 17 shows Western blots illustrating TrkA and TrkB activation forvarious compounds in cortical neurons of TrkB+/+ or −/− mice.

FIG. 18 shows Western blots illustrating deoxygedunin activation of TrkBbut not TrkA in both wild-type and TrkC knockout neurons.

FIG. 19 shows Western blots illustrating the provocation of TrkBphosphorylation by various compounds.

FIG. 20 shows Western blots illustrating the effects of variouscompounds on KA-provoked apoptosis in TrkB F616A knockin mice.

FIG. 21 shows Western blots illustrating TrkB activation by deoxygeduninin wild-type and BDNF−/− mice.

FIG. 22 shows stained inner ear sections from BDNF+/+ and −/− micetreated with vehicle or deoxygedunin (left three panels) and the rightpanel shows a graph indicating vestibular ganglion cell number indeoxygedunin (DG) and vehicle treated BDNF−/− pups.

FIGS. 23A and 23B show graphs illustrating the effect of variouscompounds on mouse immobility in forced swim tests.

FIG. 24 shows TUNEL staining images illustrating that KA provokedapoptosis is diminished by deoxygedunin in C57BL/6 mice.

FIG. 25 shows representative TTC stained brain slices 24 hours afterMCAO for vehicle-treated and deoxygedunin-treated rats.

FIG. 26 shows a tone-shock fear conditioning model developed for thesestudies.

FIG. 27 shows a graph illustrating no difference between vehicle anddeoxygedunin treated groups in shock reactivity during fear acquisitiontraining.

FIG. 28 shows a graph illustrating the difference in tone-dependentconditioned freezing on different testing days between vehicle anddeoxygedunin treated mice.

FIGS. 29A and 29B show graphs illustrating the enhanced acquisition orconsolidation of fear memory in deoxygedunin treated mice.

DETAILED DESCRIPTION

Described herein are compounds and methods for the activation of theTrkB receptor. These compounds and methods are effective in thetreatment of diseases and illnesses associated with the activation ofthe TrkB receptor including neurological disorders, neuropsychiatricdisorders, and metabolic disorders. Examples of neurological andneuropsychiatric disorders include depression, anxiety, Alzheimer's, CNSinjuries, and the like. Examples of metabolic disorders include obesityand hyperphagia. Specifically, provided herein are compounds containingfour six-membered rings and a substituted or unsubstituted C₅ or C₆heteroaryl or C₅ or C₆ heterocycloalkyl ring and pharmaceuticallyacceptable salts, prodrugs, and derivatives thereof. Methods of theiruse in the treatment of depression, anxiety, obesity, other neurologicaldisorders, and the like also are described herein.

The compounds containing four six-membered rings and a substituted orunsubstituted C₅ or C₆ heteroaryl or C₅ or C₆ heterocycloalkyl ring arerepresented by Compound I:

and pharmaceutically acceptable salts and prodrugs thereof.

In Compound I, R¹ and R² are each independently selected from hydrogen,substituted or unsubstituted C₁₋₁₂ alkyl, substituted or unsubstitutedC₁₋₁₂ haloalkyl, substituted or unsubstituted C₂₋₁₂ alkenyl, substitutedor unsubstituted C₂₋₁₂ alkynyl, substituted or unsubstituted aryl, e.g.,phenyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, substituted or unsubstituted cycloalkylalkyl, andsubstituted or unsubstituted heterocycloalkylalkyl. R¹ and R² each canbe, for example, methyl.

In Compound I, R³ is hydrogen, carbonyl, hydroxyl, —O—R¹, —O—C(═O)—R¹,or —NR⁵R⁶, wherein R⁵ and R⁶, are each independently selected from R¹.

Additionally, in Compound I, R⁴ is hydrogen, carbonyl, —R¹, —O—R¹, or—O—C(═O)—R¹. R⁴ can be, for example, —O—C(═O)—CH₃.

Also in Compound I, A is a substituted or unsubstituted C₅ or C₆heteroaryl or C₅ or C₆ heterocycloalkyl. A can be, for example,

Additionally, A can be substituted with halogen, —OR¹, or —NR⁵R⁶. Forfurther example, A can be

wherein Y¹ and Y² are each independently O, N, S, or CH₂; and Z ishydrogen, halogen, —OR⁴, or —NR⁵R⁶. Also, for example, A can be

wherein Y³, Y⁴, and Y⁵ are each independently O, N, S, or CH₂; and Z ishydrogen, halogen, —OR⁴, or —NR⁵R⁶. Further examples of A include:

Further, in Compound I, “

” is a single or double bond, wherein two double bonds are not adjacent,and “

” is a double bond or

Additional, non-limiting, examples of Compound I include:

The compounds described herein can be prepared in a variety of waysknown to one skilled in the art of organic synthesis or variationsthereon as appreciated by those skilled in the art. The compoundsdescribed herein can be prepared from readily available startingmaterials. Optimum reaction conditions may vary with the particularreactants or solvents used, but such conditions can be determined by oneskilled in the art.

Variations on Compound I include the addition, subtraction, or movementof the various constituents as described for each compound. Similarly,when one or more chiral centers is present in a molecule, the chiralityof the molecule can be changed. Additionally, compound synthesis caninvolve the protection and deprotection of various chemical groups. Theuse of protection and deprotection, and the selection of appropriateprotecting groups can be determined by one skilled in the art. Thechemistry of protecting groups can be found, for example, in Greene, etal., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons,1991, which is incorporated herein by reference in its entirety.

As used herein, the terms alkyl, alkenyl, and alkynyl include straight-and branched-chain monovalent substituents. Examples include methyl,ethyl, isobutyl, 3-butynyl, and the like. Heteroalkyl, heteroalkenyl,and heteroalkynyl are similarly defined but may contain O, S or Nheteroatoms or combinations thereof within the backbone. The termsubstituted indicates the main substituent has attached to it one ormore additional components, such as, for example, OH, halogen, or one ofthe substituents listed above.

Reactions to produce the compounds described herein can be carried outin solvents, which can be selected by one of skill in the art of organicsynthesis. Solvents can be substantially nonreactive with the startingmaterials (reactants), the intermediates, or products under theconditions at which the reactions are carried out, i.e., temperature andpressure. Reactions can be carried out in one solvent or a mixture ofmore than one solvent. Product or intermediate formation can bemonitored according to any suitable method known in the art. Forexample, product formation can be monitored by spectroscopic means, suchas nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C) infraredspectroscopy, spectrophotometry (e.g., UV-visible), or massspectrometry, or by chromatography such as high performance liquidchromatography (HPLC) or thin layer chromatography.

The methods described herein include a method of treating or reducingthe risk of disorders associated with activation of the TrkB receptorincluding neurological disorders, neuropsychiatric disorders, andmetabolic disorders in a subject. Examples of neurological andneuropsychiatric disorders include depression, anxiety, Alzheimer's, CNSinjuries, and the like. Examples of metabolic disorders include obesityand hyperphagia. This method includes the steps of selecting a subjectwith or at risk of developing the neurological disorder,neuropsychiatric disorder, or metabolic disorder, and administering tothe subject an effective amount of Compound I or derivative thereof asdescribed herein. The Compound I or derivative thereof as describedherein can be administered systemically (e.g., orally, parenterally(e.g. intravenously), intramuscularly, intraperitoneally, transdermally(e.g., by a patch), extracorporeally, topically, by inhalation,subcutaneously or the like), by administration into the central nervoussystem (e.g., into the brain (intracerebrally or intraventricularly),spinal cord, or into the cerebrospinal fluid), or any combinationthereof.

Also provided is a method of promoting neuroprotection in a subject.This method includes the steps of selecting a subject in need ofneuroprotection, and administering to the subject an effective amount ofCompound I or derivative thereof as described herein. A subject in needof neuroprotection can, for example, be a subject that has amyotrophiclateral sclerosis (ALS) or a central nervous system injury. A centralnervous system injury includes, for example, a brain injury, a spinalcord injury, or a cerebrovascular event (e.g., a stroke).

Methods can further comprise testing the effectiveness of Compound I orderivative thereof as described herein. Testing the effectiveness caninclude, but is not limited to, imaging (e.g., Magnetic ResonanceImaging (MRI)) and functional measurements (e.g., survival or clinicalsymptoms like analysis of speech patterns, logic, comprehension, memory,mood, and orientation). The method optionally further comprisesadjusting the dosage or treatment regimen of Compound I or derivativethereof as described herein.

Further provided is a method of activating a TrkB receptor on a neuron(e.g., a mammalian neuron). This method includes the steps of providinga neuron with a TrkB receptor, and contacting the TrkB receptor in vitrowith Compound I or derivative thereof as described herein in an amountsufficient to activate the TrkB receptor. Also provided is a method ofscreening for an agent that potentiates the TrkB receptor activation.The screening method includes activating the TrkB receptor on a neuronas described and contacting the neuron with the agent to be screened. Anenhanced effect indicates the agent potentiates the effect of Compound Ior derivative thereof as described herein.

The compounds described herein or derivatives thereof can be provided ina pharmaceutical composition. Depending on the intended mode ofadministration, the pharmaceutical composition can be in the form ofsolid, semi-solid or liquid dosage forms, such as, for example, tablets,suppositories, pills, capsules, powders, liquids, or suspensions,preferably in unit dosage form suitable for single administration of aprecise dosage. The compositions will include a therapeuticallyeffective amount of the compounds described herein or derivativesthereof in combination with a pharmaceutically acceptable carrier and,in addition, may include other medicinal agents, pharmaceutical agents,carriers, or diluents. By pharmaceutically acceptable is meant amaterial that is not biologically or otherwise undesirable, which can beadministered to an individual along with the selected compound withoutcausing significant unacceptable biological effects or interacting in adeleterious manner with the other components of the pharmaceuticalcomposition in which it is contained.

As used herein, the term carrier encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, orother material well known in the art for use in pharmaceuticalformulations. The choice of a carrier for use in a composition willdepend upon the intended route of administration for the composition.The preparation of pharmaceutically acceptable carriers and formulationscontaining these materials is described in, e.g., Remington'sPharmaceutical Sciences, 21st Edition, ed. University of the Sciences inPhiladelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005.Examples of physiologically acceptable carriers include buffers such asphosphate buffers, citrate buffer, and buffers with other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN® (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol(PEG), and PLURONICS™ (BASF; Florham Park, N.J.).

Compositions containing Compound I or derivative thereof as describedherein suitable for parenteral injection may comprise physiologicallyacceptable sterile aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, and sterile powders for reconstitution intosterile injectable solutions or dispersions. Examples of suitableaqueous and nonaqueous carriers, diluents, solvents or vehicles includewater, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol,and the like), suitable mixtures thereof, vegetable oils (such as oliveoil) and injectable organic esters such as ethyl oleate. Proper fluiditycan be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersions and by the use of surfactants.

Prevention of the action of microorganisms can be promoted by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, forexample, sugars, sodium chloride, and the like may also be included.Prolonged absorption of the injectable pharmaceutical form can bebrought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin.

Solid dosage forms for oral administration of Compound I or derivativethereof as described herein include capsules, tablets, pills, powders,and granules. In such solid dosage forms, the compounds described hereinor derivatives thereof is admixed with at least one inert customaryexcipient (or carrier) such as sodium citrate or dicalcium phosphate or(a) fillers or extenders, as for example, starches, lactose, sucrose,glucose, mannitol, and silicic acid, (b) binders, as for example,carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, (c) humectants, as for example, glycerol, (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates, andsodium carbonate, (e) solution retarders, as for example, paraffin, (f)absorption accelerators, as for example, quaternary ammonium compounds,(g) wetting agents, as for example, cetyl alcohol, and glycerolmonostearate, (h) adsorbents, as for example, kaolin and bentonite, and(i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules, tablets, and pills, the dosage formsmay also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethyleneglycols, andthe like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others known in the art. They may contain opacifying agentsand can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions that can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

Liquid dosage forms for oral administration of Compound I or derivativethereof as described herein include pharmaceutically acceptableemulsions, solutions, suspensions, syrups, and elixirs. In addition tothe active compounds, the liquid dosage forms may contain inert diluentscommonly used in the art, such as water or other solvents, solubilizingagents, and emulsifiers, as for example, ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils,in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil,castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol,polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures ofthese substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting, emulsifying, suspending, sweetening, flavoring, orperfuming agents. Adjuvants include, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,or mixtures of these substances, and the like.

Compositions of Compound I or derivative thereof as described herein forrectal administrations are preferably suppositories, which can beprepared by mixing the compounds with suitable non-irritating excipientsor carriers such as cocoa butter, polyethyleneglycol or a suppositorywax, which are solid at ordinary temperatures but liquid at bodytemperature and therefore, melt in the rectum or vaginal cavity andrelease the active component.

Dosage forms for topical administration of the compounds describedherein or derivatives thereof include ointments, powders, sprays, andinhalants. The compounds described herein or derivatives thereof areadmixed under sterile conditions with a physiologically acceptablecarrier and any preservatives, buffers, or propellants as may berequired. Ophthalmic formulations, ointments, powders, and solutions arealso contemplated as being within the scope of the compositions.

The term pharmaceutically acceptable salt as used herein refers to thosesalts of Compound I or derivative thereof as described herein that are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of subjects without undue toxicity, irritation,allergic response, and the like, commensurate with a reasonablebenefit/risk ratio, and effective for their intended use, as well as thezwitterionic forms, where possible, of the compounds described herein.The term salts refers to the relatively non-toxic, inorganic and organicacid addition salts of Compound I or derivative thereof as describedherein. These salts can be prepared in situ during the isolation andpurification of the compounds or by separately reacting the purifiedcompound in its free base form with a suitable organic or inorganic acidand isolating the salt thus formed. Representative salts include thehydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate,oxalate, valerate, oleate, palmitate, stearate, laurate, borate,benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate,methane sulphonate, and laurylsulphonate salts, and the like. These mayinclude cations based on the alkali and alkaline earth metals, such assodium, lithium, potassium, calcium, magnesium, and the like, as well asnon-toxic ammonium, quaternary ammonium, and amine cations including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. (See S. M. Berge et al., J. Pharm. Sci. (1977) 66:1-19,which is incorporated herein by reference in its entirety, at least, forcompositions taught herein.)

The compounds described above or derivatives thereof are useful intreating disorders associated with activation of the TrkB receptorincluding neurological disorders, neuropsychiatric disorders, andmetabolic disorders (e.g., obesity), as well as for promotingneuroprotection in humans, e.g., including pediatric and geriatricpopulations, and animals, e.g., veterinary applications. A subject inneed of neuroprotection is a subject at risk for or having a neurologicor neuropsychiatric disorder. Neurologic or neuropsychiatric disordersinclude, for example, depression, anxiety, amyotrophic later sclerosis,Alzheimer's disease, Huntington's disease, Rett syndrome, epilepsy,Parkinson's disease, and central nervous system injuries. Centralnervous system injuries include, for example, spinal cord injury,stroke, hypoxia, ischemia, and brain injury. As used herein the termspromoting, treating, and treatment includes prevention; delay in onset;diminution, eradication, or delay in exacerbation of one or more signsor symptoms after onset; and prevention of relapse.

The methods and compounds as described herein are useful for bothprophylactic and therapeutic treatment. For prophylactic use, atherapeutically effective amount of Compound I or derivative thereof asdescribed herein are administered to a subject prior to onset (e.g.,before obvious signs of neurologic or neuropsychiatric disorder), duringearly onset (e.g., upon initial signs and symptoms of neurologicaldisorder), or after an established neurological disorder. Prophylacticadministration can occur for several days to years prior to themanifestation of symptoms of a neurological or neuropsychiatricdisorder. Prophylactic administration can be used, for example, in thepreventative treatment of subjects diagnosed with genetic neurologicaldisorders such as Huntington's disease or prior to surgery in whichstroke or hypoxia is a risk. Therapeutic treatment involvesadministering to a subject a therapeutically effective amount ofCompound I or derivative thereof as described herein after a disorder,e.g., a neurological disorder, neuropsychiatric disorder, or metabolicdisorders (e.g., obesity), is diagnosed.

Administration of Compound I or derivative thereof as described hereincan be carried out using therapeutically effective amounts of Compound Ior derivative thereof as described herein for periods of time effectiveto treat neurological disorders. The effective amount of Compound I orderivative thereof as described herein may be determined by one ofordinary skill in the art and includes exemplary dosage amounts for amammal of from about 0.5 to about 100 mg/kg of body weight of activecompound per day, which may be administered in a single dose or in theform of individual divided doses, such as from 1 to 4 times per day.Alternatively, the dosage amount can be from about 0.5 to about 75 mg/kgof body weight of active compound per day, about 0.5 to about 50 mg/kgof body weight of active compound per day, about 0.5 to about 25 mg/kgof body weight of active compound per day, about 1 to about 20 mg/kg ofbody weight of active compound per day, about 1 to about 10 mg/kg ofbody weight of active compound per day, about 20 mg/kg of body weight ofactive compound per day, about 10 mg/kg of body weight of activecompound per day, or about 5 mg/kg of body weight of active compound perday. Those of skill in the art will understand that the specific doselevel and frequency of dosage for any particular subject may be variedand will depend upon a variety of factors, including the activity of thespecific compound employed, the metabolic stability and length of actionof that compound, the species, age, body weight, general health, sex anddiet of the subject, the mode and time of administration, rate ofexcretion, drug combination, and severity of the particular condition.

In these methods, the disorder being treated, e.g., depression, anxiety,central nervous system injury, obesity, or other disorder, can befurther treated with one or more additional agents. The one or moreadditional agents and Compound I or derivative thereof as describedherein can be administered in any order, including simultaneousadministration, as well as temporally spaced order of up to several daysapart. The methods may also include more than a single administration ofthe one or more additional agents and/or Compound I or derivativethereof as described herein. The administration of the one or moreadditional agent and Compound I or derivative thereof as describedherein may be by the same or different routes and concurrently orsequentially. When treating with one or more additional agents, CompoundI or derivative thereof as described herein can be combined into apharmaceutical composition with the one or more additional agents. Forexample, Compound I or derivative thereof as described herein can becombined into a pharmaceutical composition with an anti-depressant, suchas, for example imipramine, fluoxetine, paroxetine, and/or sertraline.As a further example, Compound I or derivative thereof as describedherein can be combined into a pharmaceutical composition with ananti-anxiolytic, such as, for example diazepam, alprazolam, clonazepam,and/or hydroxyzine.

The examples below are intended to further illustrate protocols forassessing the methods and compounds described herein, and are notintended to limit the scope of the claims.

EXAMPLES General Methods Cells, Reagents and Mice

For Examples 1 to 8, human neuroblastoma SH-SY5Y and human embryonickidney HEK293 cell lines are grown in DMEM with 10% fetal bovine serum(FBS) and 100 units penicillin-streptomycin at 37° C. with 5% CO₂atmosphere in a humidified incubator. Mouse septal neuron xneuroblastoma hybrids SN56 cells are created by fusing N18TG2neuroblastoma cells with murine (strain C57BL/6) neurons from postnatal21 days septa. The SN56 cells are maintained at 37° C. with 5% CO₂atmosphere in DMEM medium containing 1 mM pyruvate and 10% FBS. T48 andT62 cells, to be stably transfected with rat TrkB, are cultured in thesame medium containing 300 μg/ml G418.

For Examples 9 to 16, SN56 cells were maintained at 37° C. with 5% CO₂atmosphere in DMEM medium containing 1 mM pyruvate and 10% FBS. T48 andT62 cells, which were stably transfected with rat TrkB, were cultured inthe same medium containing 300 μg/ml G418. NGF and BDNF were from RocheDiagnostics Corporation (Indianapolis, Ind.). Phospho-Akt-473 or 308,Akt antibodies, anti-phospho-Erk1/2, and anti-phospho-TrkA Y490 werefrom Cell Signaling Technology, Inc. (Danvers, Mass.). Anti-TrkAantibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.).Anti-TrkB antibody was from BioVision, Inc. (Mountain View, Calif.).Anti-p-TrkB Y817 antibodies were from Epitomics, Inc. (Burlingame,Calif.). The chemical library containing 2000 biologically activecompounds was from the Spectrum Collection (MicroSource DiscoverySystem, Inc. Gaylordsville, Conn. 06755). TrkBF616A mice have beendescribed previously (Chen et al., 2005). TrkBF616A mice, TrkB+/−,TrkA+/− and BDNF+/−C57BL/6 mice were bred in a pathogen-freeenvironment. [³H]-Acetic acid, sodium salt (specific activity: 75-150mCi/mmol; concentration: 10 mCi/mL) was purchased from PerkinElmer, Inc.(Waltham, Mass.). Deoxygedunin was purchased from Gaia ChemicalCorporation (Gaylordsville, Conn.). All other chemicals were purchasedfrom Sigma-Aldrich Co. (St. Louis, Mo.) or Alfa Aesar (Ward Hill,Mass.).

Primary Rat Cortical Neuron Culture

Unless specifically described, primary cultured rat cortical neurons areprepared as follows. E17 rat pups are decapitated and cortex isextirpated, cross chopped, and suspended by pipetting for separation in5% fetal calf serum (FCS), 5% horse serum (HS) DMEM. The cell suspensionthen is centrifuged at 250×g for 5 minutes. This operation is repeated.Cells are seeded into polyethyleneimine-coated 10 cm² dishes and 12-wellplates including coated-coverslips and are incubated at 37° C. in 5%CO₂/95% air. After 3 hours, the culture medium is changed to Neurobasalcontaining B-27 supplement (Invitrogen; Carlsbad, Calif.) and isincubated for 4 days. For maintenance, half of the culture medium ischanged to fresh Neurobasal/B27 every 4 days. After 1 week, the dishedcultured neurons is ready for use.

Immunofluorescent Staining

Unless specifically described, primary hippocampal neurons are seeded onpoly-L lysine coated coverslips in a 12-well plate. After 7 days invitro, the neurons are treated with 100 ng/ml BDNF or variety of flavonecompounds (1 μM) for 30 minutes, and then are washed with PBS. Cells arefixed with 3% formaldehyde in PBS at room temperature for 10 minutes.The cells then are permeabilized and blocked by 0.4% Triton X-100 and 2%FBS in PBS at room temperature for 15 minutes, are washed with PBS threetimes, and are treated with anti-MAP2 (1:200) and anti-phospho-TrkBantibodies (1:500). After staining with FITC- or Rhodamine-conjugatedsecondary antibody, the coverslips are mounted on slides. Fluorescentimages are taken by a fluorescence microscope.

Immunohistochemistry Staining

Unless specifically described, brain tissues are fixed in 4%paraformaldehyde overnight followed by paraffin embedding. Sections of 5μm are cut. For immunohistochemical staining, brain sections aredeparaffinized in xylene and rehydrated in graded alcohols. Endogenousperoxidase activity are blocked by 3% hydrogen peroxide for 5 minutesand all slides are boiled in 10 mM sodium citrate buffer (pH 6.0) for 10minutes. Phosphorylated Trk A, Trk A, phosphorylated Trk B, and Trk Bare detected using specific antibodies and, e.g., a Zymed HistostainPlusAEC kit (Invitrogen; Carlsbad, Calif.). Slides then are counterstainedwith hematoxylin.

Preparation of 32-[³H]₃-Deoxygedunin.

[³H]-Acetic acid, sodium salt (11 μmol, 0.17 mL, 0.17 mL of ethanolsolution) was syringed into a heavy-walled glass vial bearing a magneticstirrer. The ethanol was removed under vacuum and replaced with 0.5 mLof THF at 0° C. Isobutylchloroformate (3.0 μL, 23 μmol) was then addedand the reaction mixture was stirred for one hour at 0° C. A solution of7-deacetyldeoxygedunin (5 mg, 11 μmol), prepared by acetyl deprotectionof deoxgedunin with K₂CO₃ in MeOH, in 0.5 mL THF was then addeddropwise. The reaction was stirred for one hour. Solvent was thenremoved under vacuum and the product was purified by preparative thinlayer chromatography (SiO₂; 1:1 EtOAc:hexanes) to give 3 mg (58%) of32-[³H]₃-deoxygedunin. Deoxygedunin was prepared under identicalreaction conditions prior to preparation of 32-[³H]₃-deoxygedunin inorder to confirm product formation.

TrkB Dimerization Assay

HEK293 cells transfected with GST-TrkB and HA-TrkA or TrkB were washedonce in PBS, and lysed in 1 ml lysis buffer (50 mM Tris, pH 7.4, 150 mMNaCl, 1 mM EDTA, 0.5% Triton X-100, 1.5 mM Na3VO4, 50 mM NaF, 10 mMsodium pyrophosphate, 10 mM sodium β-glycerophosphate, 1 mMphenylmethylsulfonyl fluoride (PMSF), 5 mg/ml aprotinin, 1 mg/mlleupeptin, 1 mg/ml pepstatin A), and centrifuged for 10 minutes at14,000×g at 4° C. The supernatant was then transferred to a fresh tubeand transfected TrkB receptor was pulled down with glutathione beads.The coprecipitated proteins were resolved on SDS-PAGE. The samples weretransferred to a nitrocellular membrane, and immunoblotting analysis wasperformed with a variety of antibodies.

Binding Constant Determination

Purified TrkB ECD or ICD proteins (10 μg/each) were incubated withdifferent [³H-deoxygedunin] in 1 ml binding buffer (0.05 M Na/Kphosphate buffer (pH 7.1), 200 mM NaCl) (1 nM [³H] deoxygedunin˜82300cpm) at 4° C. for 10 minutes. After incubation, the reaction mixture wasloaded on filter paper and washed with 3×5 ml Tris buffer (100 mM Tris,pH 7.1). The dried filter paper was put into a small vial and subjectedto liquid scintillation counter analysis. The value of the dissociationconstant and the number of sites were obtained from Scatchard plots byusing the equation r/[L]free=n/Kd−r/Kd, where r is the ratio of theconcentration of bound ligand to the total protein concentration and nis the number of binding sites.

Cortex-Specific BDNF Deletion

The Cortex-Specific Cre mouse line was previously described as“transgenic line C” (Chhatwal et al., Gene Ther. 14, 575-583 (2007)).Briefly, coding sequence for Cre-recombinase (Cre-IRES-DsRed2) wasplaced downstream of a 3 kb cholecystokinin (CCK) promoter, linearized,purified, and microinjected into the pronuclei of one-cell C57/BL6embryos, which were then implanted into pseudopregnant C57/BL6 females.Following verification of gene expression in the different transgeniclines (Chhatwal et al., Nat. Neurosci. 9, 870-872 (2006)), thecortex-specific “line C” was crossed to a floxed-stop lacZ reportermouse line (Soriano, Nat. Genet., 21, 70-71 (1999)) as well as thefloxed BDNF mouse line (Rios et al., Mol. Endocrinol., 15, 1748-1757(2001)). Region specific Cre gene expression and BDNF deletion wereconfirmed with in situ hybridization, x-gal staining for β-galactosidaseexpression, and Western blot for BDNF protein levels.

Vestibular Ganglion Dissection in BDNF−/− Pups

The cochleae of various drug-treated pups (P1 or P2 BDNF+/+ and −/−pups) were first fixed through cardioperfusion of 4% paraformaldehyde(in PBS). Each cochlea was dissected out and postfixed in 1% osmium forone hour at room temperature. Samples were decalcified in 0.35 M EDTA(pH 7.5, in PBS) for 72 hours at 4° C., followed by gradual dehydrationin graded alcohols, infiltrated, and embedded in epoxy resin with theconventional protocols. Consecutive cochlear sections (5 μm inthickness) were cut with a microtome (Microm HM335E, GmbH) along theaxis of the modiolus. Sections were stained with toluidine blue.Vestibular ganglions were identified by their location in the auditoryinternal meatus with the basal cochlear turn and the cochlear modiolusas morphological reference landmarks.

Focal Ischemia Model

A total of 12 rats were used (1 rat was excluded because of inadequatereperfusion). Focal cerebral ischemia was induced by occlusion of theright middle cerebral artery as described by Sayeed et al., Ann. Emerg.Med. 47, 381-389 (2006). Drug Administration: The rats subjected to MCAOincurring ischemic insult <40% of baseline LDF were randomly assigned toreceive either deoxygedunin (n=4), 7,8-DHF (n=4), or vehicle (n=4)treatment. Deoxygedunin and 7,8-DHF were given at the dose of 5 mg/kg byi.p. injection 5 minutes prior to the onset of reperfusion. Rats in thevehicle group underwent the same experimental protocol, except that theyreceived an identical volume/weight of vehicle only. Statisticalanalysis: All results are expressed as mean±S,E,M. Mean ischemic lesionvolume were analyzed using the Student's t-test. The criterion forstatistical significance was set at p<0.05.

Mouse Conditioned Fear Studies

Following a two-day habituation to testing context, wild-type C57Bl/6Jmice (N=28, male, 8-10 weeks old) were fear conditioned in eightidentical startle response systems (SR-LAB, SDI) consisting of anonrestrictive Plexiglas cylinder, 5.5 cm in diameter and 13 cm long,mounted on a Plexiglas platform which was located in a ventilated,sound-attenuated chamber. One hour prior to fear conditioning, micereceived 8-OH-Deoxygedunin (N=14, 5 mg/kg, i.p.) or vehicle (N=14, 17%DMSO in PBS). Mice then received 5 tone-footshock pairings, with 30second 12 kHz, 85 dB tones which co-terminated with the footshocks(intensity of 0.5 mA, 0.5 second) with a 5 minute intertrial interval(ITI), after which they were returned to their homecage. 24 and 48 hrsafter training, the mice were tested for freezing in rodent modular testchambers with an inside area of 30.5 cm×24.1 cm×21.0 cm. Three minutesafter placing the mouse in the test chamber, fifteen 30 secondconditioned stimulus (CS) tones with an ITI of 1.5 min were deliveredthrough a high-frequency speaker attached to the side of each chamber.Percentage time spent freezing during the CS presentations wascalculated for each mouse using FreezeFrame (Product Number: ACT-100)(Coulbourn Instruments, Whitehall, Pa.).

Example 1 Cell-Based Screen to Identify Compounds that Protect TrkBExpressing Cells from Apoptosis

To Create and Test Reporter Cell Lines.

In order to identify small molecules that mimic BDNF and activate TrkB,TrkB stably transfected murine cell lines are created. The T48 and T62cell lines are created by transfecting basal forebrain SN56 cells, whichexpress negligible TrkB, with a TrkB expression construct. To testexpression of TrkB, the cells are treated with BDNF, which is predictedto result in strong phosphorylation of Trk-490 and Akt activation incomparison to the TrkA NTR stably expressing T17 cell line indicatingexpression of TrkB. To test resistance to apoptosis, the SN56 cells andthe T48 cell line are either untreated or treated with BDNF, and thenare subjected to an apoptotic assay. The apoptotic assay involvestreating the cells with 0.75 μM Staurosporine for 9 hours, and 1 hourbefore completing the experiment, the cells are treated with 10 μM MR(DEVD)2. The cells then are fixed with 4% paraformaldehyde for 15minutes, washed with phosphate buffered saline (PBS), and incubated withHoechst 33342 for 10 minutes. Cover slides are washed with PBS, mounted,and then the cells are examined using a fluorescent microscope to seewhich cells turn red upon caspase cleavage.

Cell-Based Screen.

To screen Compound I and derivatives thereof, a cell-based apoptic assayis used. The screen employs a cell permeable fluorescent dye, MR(DEVD)2, which turns red upon caspase cleavage in apoptotic cells. SN56and T48 cells, which are created as described above, are plated at10,000 cells per well in multiple 96-well plates and are exposed toCompound I and derivatives thereof for 30 minutes at a concentration of10 μM in DMSO. Following exposure to the compounds, the cells aresubjected to the developed fluorescent apoptotic assay described above(method schematically shown in FIG. 1).

Candidates that selectively protect the T48 cell line, but not the SN56cell line, then are subjected to a neurite outgrowth assay of SH-SY5Ycells for a secondary screen. Any positive compounds, i.e., identifiedactive compounds, are further validated for TrkB activation, PI-3kinase/Akt and MAP kinases signaling cascade activation in primaryhippocampal neurons.

Example 2 Identification of Survival Enhancers

To compare the apoptosis inhibitory activity of identified activecompounds, the compounds are preincubated with SN56 and T48 cells, andsubsequently are subjected to the fluorescent apoptotic assay asdescribed above. To examine whether these identified active compoundspromote neuronal survival, hippocampal neurons are prepared and thecultures are pretreated with the identified active compounds for 30minutes, followed by treatment with 50 μM glutamate for 16 hours. Aquantitative apoptosis assay, for example, demonstrates theeffectiveness of any active compounds.

To explore whether the identified active compounds exert a protectiveeffect on hippocampal neurons in Oxygen-Glucose Deprivation (OGD),primary preparations of neurons are treated with BDNF or various flavonederivatives for 30 minutes prior to OGD. After 3 hours, apoptoticanalysis demonstrates whether an active compound has a protectiveeffect. Further, a titration assay reveals whether an active compoundprotects neurons in a dose-dependent manner.

Example 3 Protocol to Determine Whether an Identified Active CompoundTriggers TrkB Activation in Hippocampal Neurons In Vitro

BDNF binding to TrkB induces its autophosphorylation and, subsequently,activation of downstream kinase pathways including MAPK and PI3/Akt. Toexplore whether an identified active compound triggers TrkB activation,immunofluorescent staining on hippocampal neurons with anti-phospho TrkBantibody is conducted. To examine whether an identified active compoundstimulates TrkB-mediated downstream signaling cascades, Western analysisis performed and the activation of Akt and Erk also is monitored. Totest whether the stimulatory effect of an identified active compound ismediated through TrkB, cells are either untreated or treated with K252a,a selective inhibitor of the tyrosine kinase activity of the Trk familyof neutrophin receptors. Cells treated with K252a block TrkB tyrosinephosphorylation in cells exposed to an identified active compound. Toprobe the time course of TrkB activation triggered by an identifiedactive compound, hippocampal neurons are treated with an identifiedactive compound at 500 nM and phosphorylation of Erk and Akt isdetermined over time by Western analysis. Whether stimulation of Erk andAkt by an identified active compound occurs in a dose dependent manneralso is determined.

Example 4 Protocol to Determine Whether an Identified Active CompoundTriggers TrkB Activation in Hippocampal Neurons In Vivo

To assess whether an identified active compound provokes TrkB activationin the brain, mice are intraperitoneally injected with a dose of 5 mg/kgand analyzed at various time points. Western analysis reveals whetherTrkB, but not TrkA, is selectively phosphorylated in the brain afterinjection. Further, whether the protein and mRNA levels of theneurotrophic receptors is altered after treatment with an identifiedactive compound is measured. Immunofluorescent staining of the braindisplays substantial TrkB phosphorylation in the hippocampus for anactive compound.

Example 5 Protocol to Determine Whether an Identified Active CompoundBinds the Extracellular Domain of the TrkB Receptor

BDNF is known to bind the TrkB receptor and provoke its dimerization(Barbacid, J. Neurobiol., 25:1386-1403, 1994; Klein et al, Cell,66:395-403, 1991). To explore whether an identified active compoundtriggers TrkB receptor dimerization, HEK293 cells are cotransfected withGST-TrkB and HA-TrkB or HA-TrkA. The cells then are treated with BDNF oran identified active compound (0.5 μM) for 30 minutes. The cells thenare harvested, washed once in PBS, and lysed in 1 ml lysis buffer (50 mMTris, ph 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1.5 mM Na₃VO₄,50 mM NaF, 10 mM sodium pyrophosphate, 10 mM sodium β-glycerophosphate,1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mg/ml aprotinin, 1 mg/mlleupeptin, 1 mg/ml pepstatin A) and are centrifuged for 10 minutes at14,000×g at 4° C. The supernatant is transferred to a fresh tube andtransfected TrkB receptor is separated from the supernatant withglutathione beads, and the coprecipitated proteins are resolved onSDS-PAGE. The samples are transferred to nitrocellular membrane, andWestern analysis demonstrates whether the identified active compoundprovoked TrkB dimerization to a similar manner as BDNF.

To determine if an identified active compound promotes tyrosinephosphorylation of the other Trk receptors, HEK293 cells are transfectedwith various Trk receptors, which is followed by treatment with theidentified active compound. Treatment with an identified active compoundthat elicits tyrosine phosphorylation in the TrkB receptor but not inthe TrkA or TrkC receptor indicates an active compound.

To determine whether an identified active compound physically anddirectly binds to the TrkB receptor, in vitro binding assays can beconducted with purified TrkB extracellular domain (ECD) andintracellular domain (ICD) recombinant proteins. Purified TrkB ECD andICD (10 μg of each) are incubated with different concentrations of³H-labeled identified active compound in 1 ml of binding buffer (0.05MNa/K phosphate buffer, pH 7.1, 200 mM NaCl) at 4° C. for 10 minutes.After the incubation, the reaction mixture is loaded on filter paper.The mixture is washed three times with Tris buffer (100 mM Tris, pH7.1), and the dried filter paper is put into a small vial and subjectedto liquid scintillation counter analysis. Gradual increments of[³H]-labeled identified active compound indicate progressively boundTrkB ECD but not ICD. The value of the dissociate constant and thenumber of sites will then be obtained from Scatchard plots using theequation r/[L]_(free)=n/K_(d)−r/K_(d), where r is the ratio of theconcentration of bound ligand to the total protein concentration and nis the number of binding sites. Quantitative analysis using theScatchard plot reveals whether the ratio of ligand to the receptor is1:1 and the binding constant K_(d).

To further explore the association of an identified active compound andthe TrkB receptor, an in vitro binding assay can be performed.Increasing volumes of GST-TrkB ECD and GST-TrkB ICD are bound toglutathione beads to a total of 250 uL, and 500 nM identified activecompound in 250 μl (20% DMSO/80% PBS) are incubated with the beads inthe column at 4° C. for 30 minutes. After the incubation, the elutefractions are collected and the concentration of eluted identifiedactive compound is analyzed by UV-spectrometry at a wavelength of 410nm.

BDNF is known to bind to the region of the TrkB ECD that contains thethird leucine-rich motif (LRM), the second cysteine cluster (CC) domain,and the Immunoglobulin 2 (Ig2) domain (Haniu et al., J. Biol. Chem.,272:25296-303, 1997). To map where an identified active compound bindson the TrkB ECD, truncation mutants of the ECD is made and in vitrobinding assays are conducted. From association data obtained from thetruncated mutants, the binding regions are determined.

Example 6 Protocol to Determine Whether an Identified Active CompoundPrevents Kainic Acid-Triggered Neuronal Apoptosis and Decreases InfarctVolume of Stroked Rat Brain

Kainic acid (KA) is a potent agonist for the AMPA receptor. Peripheralinjections of KA result in recurrent seizures and the subsequentdegeneration of select populations of neurons in the hippocampus(Schauwecker and Steward, Proc. Natl. Acad. Sci. USA, 94:4103-8, 1997).KA induces neuronal cell death in a caspase-dependent and independentmanners (Faherty et al., Brain Res. Mol. Brain Res., 70:159-63, 1999;Glassford et al., Neurol. Res., 24:796-800, 2002; Liu et al., Mol. Cell,29:665-78, 2008). To explore whether an identified active compoundblocks the neurotoxicity initiated by KA, C57BL/6 mice aged 60 days areintraperitoneally injected with either a single dose of 20% DMSO insaline, 20 mg/kg KA, or 5 mg/kg of an identified active compoundfollowed by 20 mg/kg of KA. After 5 days, the mice are anesthetized,perfused with 4% paraformaldehyde in 0.1M phosphate buffered saline, andthe brains are removed, post-fixed overnight, and processed for paraffinembedding. Serial sections of the brain are cut to a thickness of 5 μmand mounted on slides. TUNEL staining reveals whether KA provokesapoptosis in the hippocampus, which is diminished by an active compound.

To further determine the neuroprotective potential in vivo, anidentified active compound can be tested in a transient middle cerebralartery occlusion (MCAO) stroke model in adult male rats. Focal cerebralischemia is induced by occlusion of the right middle cerebral artery aspreviously described (Sayeed et al, Ann. Emerg. Med., 47:381-9, 2006).After 2 hours MCAO followed by reperfusion, the animals receive vehicleor an identified active compound (5 mg/kg) intraperitoneally 5 minutesprior to the onset of reperfusion. Survival of the ischemic insult aftertreatment with an identified active compound demonstratesneuroprotection. Further, brain slices stained with 2, 3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO invehicle-treated and identified active compound-treated rats indicate aneuroprotective effect.

Example 7 Protocol to Determine Whether an Identified Active CompoundProtects Neurons from Apoptosis in TrkB Dependent Manner

To determine whether an identified active compound selectively activatesTrkB receptor and prevents neuronal cell death, cortical neurons areprepared from homozygous pups of TrkB+/− mice, which are mated to thesame genotype. The activation of TrkB and down stream indicators of TrkBactivation, such as Capase-3, are monitored. To further assess whetheran identified active compound blocks neuronal apoptosis in a TrkBdependent manner, cortical neurons are prepared from homozygous pups ofTrkC+/− mice, which are mated to the same genotype. Again, theactivation of TrkB and down stream indicators of TrkB activation, suchas Capase-3, are monitored.

To explore whether the neuroprotective action of an identified activecompound is dependent on TrkB activation in vivo, TrkB F616A knockinmice are used. The TrkB F616A receptor has been shown to be selectivelyblocked by 1NMPP1 inhibitor and lead to TrkB-null phenotypes (Chen etal., Neuron, 46:13-21, 2005). To further assess whether an identifiedactive compound can mimic BDNF, cortical neurons are prepared from TrkBF616A knockin mice. The cortical neurons are pretreated for 30 minuteswith either K252a Trk tyrosine kinase inhibitor (100 nM) or 1NMPP1inhibitor (100 nM) followed by 0.5 μM identified active compound for 30minutes. Whether TrkB phosphorylation is selectively blocked by 1NMPP1,is monitored.

To determine if 1NMPP1 makes neurons treated with an identified activecompound vulnerable to KA-provoked neuronal cell death, TrkF616A knockinmice are fed with 1NMPP1 (25 mM) in drinking water one day prior topharmacological reagent treatment. The next day, the mice areintraperitoneally injected with KA (25 mg/kg), or an identified activecompound (5 mg/kg) 4 hours prior to KA injection. The control mice areinjected with either KA or an identified active compound alone, or themice are administered an identified active compound 4 hours before KA.After 4 days, the mice are sacrificed and the brains are homogenized andultracentrifuged. The supernatant then is employed for SDS-PAGE andimmunoblotting analysis. Whether the identified active compoundsuppresses KA-provoked apoptosis, i.e., exhibits a neuroprotectiveeffect, is determined.

Example 8 Protocol to Determine Whether an Identified Active CompoundDisplays an Anti-Depressive Effect

BDNF has been shown to play an essential role in mediatingantidepressants' therapeutic effects (Casten, Curr. Opin. Pharmacol.,4:58-64, 2004; Duman, Biol.

Psychiatry, 56:140-5, 2004; Groves, Mol. Psychiatry, 12:1079-88, 2007;Monteggia et al., Proc. Natl. Acad. Sci. USA, 101:10827-32, 2004;Saarelainen et al., J. Steroid Biochem. Mol. Biol., 78:231-9, 2003).Further, infusion of exogenous BDNF into hippocampus or brain stem hasbeen shown to have an anti-depressant-like behavioral effect (Shirayamaet al., J. Neurosci., 22:3251-61, 2002; Siuciak et al., Pharmacol.Biochem. Behav., 56:131-7, 1997). To explore whether an identifiedactive compound has an antidepressant effect like BDNF, a forced swimtest is conducted. Adult male mice (2-3 months old) are randomlysubmitted, without a pre-swim, to a forced swim test of 6 minutes withimmobility recorded in the last 4 minutes. The mice are injectedintraperitoneally for 5 days with saline, imipramine (20 mg/kg),amitryptyline (15 mg/kg), or an identified active compound (5 mg/kg).The mice are allowed to adapt to the test room for 2 days, and the miceare placed in a clear glass cylinder with a diameter of 16 cm,half-filled with clear water at 24° C. The water depth of 14 cm does notallow the mice to reach the bottom of the cylinder, and the water ischanged after each mouse. Mice treated with an identified activecompound exhibiting an anti-depressive effect show increased mobility.

Example 9 Identification of Gedunin Derivatives as Survival Enhancers

Using a cell-based screen as described above in Example 1 (based on theanti-apoptotic action of TrkB signaling) and the chemical library fromthe Spectrum Collection (described above), 66 positive hits weregenerated, four of which were gedunin derivatives. The SpectrumCollection library also contained numerous gedunin derivatives, whichdid not generate hits. The chemical structures of 12 gedunin relatedcompounds contained in the Spectrum Collection library (including bothhits and non-hits) are shown in FIG. 2. To compare apoptosis inhibitoryactivity, each of these compounds (0.5 μM) was preincubated withhippocampal neurons for 30 min, followed by 50 μM glutamate for 16 h.Among the 12 gedunin derivatives (see FIG. 3), deoxygedunin (I-33)displayed the most robust protective effect, followed byalphadihydrogedunol (epoxy ring down) (I-30) and dihydrodeoxygedunin(I-16). In this assay, neurons with both cleaved caspase (MR(DEVD)2 redcells) and condensed nuclei (DAPI staining) were counted as apoptoticcells. As discussed above in Example 2, OGD (Oxygen-Glucose Deprivation)was used an in vitro model for ischemic stroke. Apoptotic ratio wascompared to neurons treated with BDNF which was known to reduce ischemicinjury (Kurozumi et al., 2004; Schabitz et al., 2000) and DMSO which hasno protective effect. Deoxygedunin, alpha-dihydrogedunol (epoxy ringdown) and dihydrodeoxygedunin exhibited potent protective effects onhippocampal neurons under OGD (see FIG. 4, left panel). As shown in FIG.4, right panel, a titration assay shows that deoxygedunin protectsneurons in a dose-dependent manner.

Example 10 Deoxygedunin Activates TrkB and Protects Neurons fromApoptosis

Deoxygedunin (among others) elicited a strong TrkB phosphorylation (seeFIG. 5), which was also independently confirmed by immunoblottinganalysis (see FIG. 6). Both Akt and Erk1/2 were robustly activated bythese molecules as well). In hippocampal neurons, deoxygeduninprominently provoked both Erk1/2 and Akt activation with a time course(see FIG. 7, left panels) and stimulated both Erk1/2 and Akt activationin a dose dependent manner (see FIG. 7, right panels). The minimalrequired drug concentration was about 100-250 nM (see FIG. 7, rightpanels). These data demonstrate that deoxygedunin potently activatedTrkB receptor and its downstream Akt and MAP kinases in neurons. Tofurther demonstrate that deoxygedunin activates TrkB receptor,hippocampal neurons were pretreated with K252a (a Trk receptorsinhibitor) and TrkB activation was examined (see FIG. 8). As shown inFIG. 8, pretreatment with K252a substantially blockeddeoxygedunin-triggered TrkB activation in hippocampal neurons,demonstrating that deoxygedunin can provoke TrkB autophosphorylation.Deoxygedunin-provoked downstream Akt and MAPK signalings were alsoblocked by K252a. To assess whether deoxygedunin provokes TrkBactivation in the brain, mice (i.p.) were injected with a dose of 5mg/kg for various time points. TrkB but not TrkA was selectivelyphosphorylated in the brain 2 hours after injection, and peaked at 4-8hours (see FIG. 9), suggesting that deoxygedunin penetrated theblood-brain barrier and stimulated TrkB activation. To establish thatdeoxygedunin is orally bioactive in provoking TrkB activation, two tothree month old C57BL/6J mice were orally injected with various doses of7,8-dihydroxyflavone or deoxygedunin then sacrificed 2 to 4 hours afteradministration. Brain lysates were prepared and analyzed byimmunoblotting (see FIG. 10). FIG. 10 shows that TrkB was orallyactivated in mouse brain by both 8-dihydroxyflavone and deoxygeduninwith dosages as low as 1-5 mg/kg. RT-PCR analysis revealed no change ofTrkA or TrkB in mouse brain upon deoxygedunin treatment, indicating thatdeoxygedunin provokes TrkB activation independent of Trk receptortranscriptional alteration. Immunohistochemistry staining demonstratedrobust TrkB activation in hippocampus upon deoxygedunin treatment (seeFIG. 11). These data demonstrate that deoxygedunin strongly triggeredTrkB activation both in vitro and in vivo.

Example 11 Deoxygedunin Binds TrkB ECD and Provokes its Dimerization

To determine whether deoxygedunin binds the intra-cellular domain (ICD)or extra-cellular domain (ECD) of the TrkB receptor, ligand bindingassays with [³H]-deoxygedunin were performed. The assays demonstratedthat increasing concentrations of [³H]-deoxygedunin progressively boundthe TrkB extra-cellular domain (ECD) but not the intra-cellular domain(ICD) (see FIGS. 12A and B). Additionally, [³H]-deoxygedunin did notbind to TrkA, indicating it specifically associated with theextracellular domain of TrkB receptor (see FIG. 12A). Truncation assaysshowed that the Ig2 domain in the ECD of TrkB was the major binding sitefor deoxygedunin (FIG. 12B). Scatchard plot analysis revealed that theratio of ligand to the receptor is 1:1 with binding constant Kd=1.4 μM(FIG. 13). A GST pull-down assay revealed that deoxygedunin robustlyprovoked TrkB dimerization with an effect even stronger than BDNF (seeFIG. 14). Moreover, alpha-dihydrogedunol (epoxy ring down) also notablypromoted TrkB dimerization (see FIG. 14), fitting with its stimulatoryactivity on TrkB (see Example 10). The coprecipitated HA-TrkB was alsoprominently tyrosine phosphorylated (see FIG. 14, 3^(rd) panel). Thesedata indicated that deoxygedunin directly bound TrkB ECD and triggeredits association. Truncation assays showed that deletion of Ig2 domain inTrkB diminished its association by deoxygedunin (FIG. 15). Deoxygeduninalso elicited tyrosine phosphorylation in TrkB but not in TrkA or TrkCreceptor in transfected HEK293 cells. TrkB-KD displayed negligiblephosphorylation compared to wild-type TrkB (FIG. 16), indicating thatTrkB phosphorylation by deoxygedunin was through the receptorautophosphorylation but not by any other tyrosine kinases. These datashowed that deoxygedunin bound to the ECD of TrkB and promoted itsassociation and activation.

Example 12 Deoxygedunin Protects Neurons from Apoptosis in aTrkB-Dependent Manner

To determine if deoxygedunin's neuronal protective effect was mediatedthrough TrkB receptor, cortical neurons from pups of TrkB+/− mice matedto the same genotype were prepared. Deoxygedunin specifically activatedTrkB but not TrkA receptor in wild-type but not TrkB−/− neurons.7,8-dihydroxyflavone (7,8-DHF), another positive compound from thescreening, also selectively activated TrkB but not TrkA. The tricyclicantidepressant drugs amitriptyline but not imipramine activated bothTrkA and TrkB (FIG. 17, top and 3rd panels). Glutamate-provokedcaspase-3 activation was substantially blocked by 7,8-DHF anddeoxygedunin in wild-type but not TrkB−/− neurons. However, the controlcompound imipramine failed to block caspase-3 activation by glutamate.In contrast, amitriptyline weakly suppressed caspase-3 activation inboth wild-type and TrkB−/− neurons (FIG. 17, bottom panels). Thus,deoxygedunin selectively suppressed apoptosis triggered by glutamate ina TrkB dependent manner. Moreover, deoxygedunin strongly provoked TrkBbut not TrkA activation in both wild-type and TrkC knockout neurons(FIG. 18, top panel). Additionally, the spontaneous caspase-3 activationin TrkC−/− neurons was suppressed by deoxygedunin. Further, glutamatetriggered caspase-3 activation was diminished by deoxygedunin (FIG. 18,bottom panel), indicating that it repressed neuronal apoptosis in aTrkB- but not TrkC-dependent.

TrkB F616A was known to be selectively blocked by 1NMPP1 resulting in aneffective TrkB-null phenotype (see Chen et al., Neuron 46, 13-21(2005)). BDNF-provoked TrkB phosphorylation was selectively blocked by1NMPP1 but not K252a in cortical neurons from TrkB F616A knockin mice(see FIG. 19, top panel). Similarly, deoxygedunin provoked TrkBphosphorylation was selectively blocked by 1NMPP1 but not K252a incortical neurons from TrkB F616A knockin mice (FIG. 19, top panel).Additionally, 1NMPP1, but not K252a, blocked BDNF-triggered Akt andErk1/2 activation. Similarly, 1NMPP1 diminished Akt and Erk1/2activation by deoxygedunin (FIG. 19, 3rd and 5th panels). 1NMPP1'sselective inhibition of TrkB F616A activation by deoxygedunin, suggestedthat the blockade of TrkB F616A signaling by 1NMPP1 in mice makes theneurons vulnerable to KA-provoked neuronal cell death. KA causedcaspase-3 activation, and pretreatment of 1NMPP1 elevated KA-provokedapoptosis in TrkB F616A (see FIG. 20), supporting that TrkB signalingwas involved in neuronal survival. Deoxygedunin suppressed KA-provokedapoptosis, whereas 1NMPP1 pretreatment diminished deoxygedunin'sprotective effect in F616A mice. TrkB activation status inverselycorrelated with TrkB activation by deoxygedunin (FIG. 20, top and middlepanels). These data show that deoxygedunin selectively activated TrkBreceptor and enhanced neuronal survival in mice in TrkB dependentmanner.

Example 13 Deoxygedunin Activates TrkB in BDNF Independent Manner andPrevents Vestibular Ganglion Loss

To examine whether deoxygedunin activating TrkB involved endogenousBDNF, BDNF conditional knockout mice with BDNF gene deletion limited tocortex (thus allowing normal development) were used. Deoxygedunin (5mg/kg) was intraperitoneally injected into the BDNF cortex conditionalknockout mice and the mice were sacrificed at 4 hours. TrkB activationoccurred in both wild-type and BDNF−/− mice (see FIG. 21), demonstratingthat deoxygedunin activated TrkB independent of BDNF. Mutant micelacking BDNF were known to have severe deficiencies in coordination andbalance, which have been associated with excessive degeneration inseveral sensory ganglia including the vestibular ganglion (Ernfors etal., Nature 368, 147-150 (1994)). To determine whether deoxygeduninrescued this loss of vestibular ganglions in BDNF−/− pups, conventionalBDNF+/− mice were bred with the same genotype mice. Deoxygedunin (5mg/kg, i.p.) was administered to the pregnant mice at day E7.5 untilbirth. The neonatal pups continued receiving the same dose ofdeoxygedunin, but BDNF−/− pups continued dying at P1 or P2. Staining ofinner ear sections showed that vestibular ganglia were completely lostin most of control vehicle-treated BDNF−/− pups. In contrast, many ofdeoxygedunin-treated BDNF mutant mice displayed intact vestibularganglia, similar to the wild-type pups (see FIG. 22, left three panels).Quantitative analysis demonstrated that 9.1±4.9% of vestibular gangliawere detected in vehicle-treated BDNF−/− pups, whereas deoxygedunintreatment increased to 42.2±6.3% (see FIG. 22, right panel). These datashow that deoxygedunin mimicked BDNF and significantly protectedvestibular ganglia from degeneration in BDNF−/− pups.

Example 14 Deoxygedunin has an Antidepressant Effect

To investigate whether deoxygedunin mimicked BDNF in suppressingdepression-like symptoms, a forced swim test after subchronic treatmentof the mice for 5 days with various drugs was conducted. When mice weretreated with imipramine (20 mg/kg), a tricyclic antidepressant drug, theswimming immobility was significantly decreased. Deoxygedunin (5 mg/kg)also reduced the immobility (see FIG. 23A). To assess whether thebehavior responses by 7,8-DHF and deoxygedunin were mediated by TrkBreceptor, TrkB F616A knockin mice were used. The transgenic mice weresubjected to saline or 1NMPP1 treatment, respectively. No significantdifference was observed in the immobility time between saline and1NMPP1-treated mice. In the saline group, deoxygedunin substantiallyreduced the immobility time; however, deoxygedunin did not have asignificant effect in mice when TrkB was blocked by 1NMPP1 (see FIG.23B), suggesting that inhibition of the TrkB signaling cascade inhibitedthe antidepressant effect of deoxygedunin. Thus, these data show thatdeoxydegunin mimicked BDNF and acted as a potent antidepressant drug inmice through activating the TrkB receptor.

Example 15 Deoxygedunin Displays Therapeutic Effects on VariousNeurological Disorders

Kainic acid (KA), a specific agonist for the kainate receptor, was knownto induce neuronal cell death in caspase-dependent and independentmanners. To explore whether deoxygedunin can block the neurotoxicityinitiated by KA, 5 mg/kg deoxygedunin was intraperitoneally injectedinto C57BL/6 mice, followed by 20 mg/kg KA. In 5 days, the mice wereperfused and the brains were cut to a thickness of 5 μm and mounted onslides. TUNEL staining revealed that KA provoked apoptosis in thehippocampus, but that apoptosis was diminished by deoxygedunin (FIG. 24,left panel). Quantitative analysis of apoptosis in the hippocampusrevealed that deoxygedunin decreased KA induced apoptosis in hippocampusby 60% (FIG. 24, right panel). To further determine the neuroprotectivepotential in vivo, deoxygedunin was tested in a transient middlecerebral artery occlusion (MCAO) stroke model in adult male rats. After2 h MCAO followed by reperfusion, the animals received vehicle ordeoxygedunin (5 mg/kg) 5 minutes prior to the onset of reperfusion. Allanimals included in the study survived the ischemic insult and treatmentwith deoxygedunin. Representative brain slices stained with TTC 24 hoursafter MCAO for vehicle-treated and deoxygedunin-treated rats are shownin FIG. 25 (left panel). Area and volume measurements from the TTCstained sections indicated that treatments with deoxygeduninsignificantly reduced infarct volumes in this transient ischemic modelof stroke (FIG. 25, right panel). These results indicated thatpretreatment with intraventricular BDNF reduced infarct size after focalcerebral ischemia in rats and supported the hypothesis of aneuroprotective role for BDNF in stoke. Taken together, these dataindicated that deoxygedunin prevented neuronal cell death and wasprotective of the neurodegeneration elicited by excitatory neurotoxicityand stroke.

Example 16 Deoxygedunin Enhances Acquisition of Conditioned Fear, aBDNF-Dependent Learning Process

To determine whether deoxygeduning would enhance learning in a wholeanimal model of learning and memory, in which BDNF-dependent TrkBactivation was required, a tone-shock fear conditioning model wasdeveloped (see FIG. 26). Following habituation to the testing context,28 adult wild-type, C57BL/6J mice were given systemic injections ofdeoxygedunin (N=14, 5 mg/kg, i.p.) or vehicle (N=14) 1 hour prior tobeing subjected to the tone-shock fear conditioning model. There was nodifference between treatment groups in shock reactivity during the fearacquisition training, suggesting that there were no acute effects onpain sensitivity that would affect fear acquisition or later fearexpression (p>0.1; see FIG. 27). Mice were then tested, with noadditional drug treatment, for cue-conditioned fear in the previouslyhabituated context on the two days following fear acquisition. Theaverage level of tone-dependent conditioned freezing was significantlydifferent on both testing days (see FIG. 28; repeated measures ANOVA,F(1,26)=6.6, p=0.016) suggesting that mice that received deoxygedunin atthe time of training had enhanced acquisition or consolidation of thefear memory. To further explore these effects, individual animals'freezing levels throughout the tone-fear testing sessions were examined.On both testing day 1 (see FIG. 29A) and day 2 (see FIG. 29B), theenhancement in freezing only corresponded with the periods of tone cuepresentation. The mice demonstrated similar levels of locomotorexploratory activity prior to and in-between tone exposure in thiscontext, but the animals that received deoxygedunin during the previoustoneshock fear conditioning demonstrated increased freezing during cuedfear presentations (Day 1, repeated measures ANOVA of first 4 CS trials,F(1,26)=8.1, p<0.01; Day 2, F(1,26)=7.5, p<0.01). This increase in fearlearning led to a 2-3 fold increase in the level of freezing during thefirst set of conditioned stimulus (CS) trials examined each day.Together, these results suggested that although deoxygedunin neitheraffected apparent level of pain or shock reactivity during training noraffected general locomotor activity in the testing situation onsubsequent days, the learning event that occurred during training in thepresence of systemic deoxygedunin compared with vehicle was acquired orconsolidated in a more effective manner. Since cue-dependent fearconditioning was known to require, and be exquisitely sensitive to, BDNFactivation of TrkB, these data were consistent with deoxygedunin actingon the TrkB system in vivo to enhance cue-dependent fear learning.

The compounds and methods of the appended claims are not limited inscope by the specific compounds and methods described herein, which areintended as illustrations of a few aspects of the claims and anycompounds and methods that are functionally equivalent are within thescope of this disclosure. Various modifications of the compounds andmethods in addition to those shown and described herein are intended tofall within the scope of the appended claims. Further, while onlycertain representative compounds, methods, and aspects of thesecompounds and methods are specifically described, other compounds andmethods and combinations of various features of the compounds andmethods are intended to fall within the scope of the appended claims,even if not specifically recited. Thus a combination of steps, elements,components, or constituents may be explicitly mentioned herein; however,all other combinations of steps, elements, components, and constituentsare included, even though not explicitly stated.

What is claimed is:
 1. A method of treating Alzheimer's diseasecomprising administering to a subject in need thereof an effectiveamount of a pharmaceutical composition comprising a compound of thefollowing formula:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: R¹and R² are each independently selected from hydrogen, substituted orunsubstituted C₁₋₁₂ alkyl, substituted or unsubstituted C₁₋₁₂ haloalkyl,substituted or unsubstituted C₂₋₁₂ alkenyl, substituted or unsubstitutedC₂₋₁₂ alkynyl, substituted or unsubstituted aryl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heteroaryl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl,substituted or unsubstituted cycloalkylalkyl, and substituted orunsubstituted heterocycloalkylalkyl; R³ is hydrogen, carbonyl, hydroxyl,—O—R¹, —O—(C═O)—R¹, or —NR⁵R⁶, wherein R⁵ and R⁶, are each independentlyselected from R¹; R⁴ is carbonyl, —R¹, —O—R¹, or —O—(C═O)—R¹; A is asubstituted or unsubstituted C₅ or C₆ heteroaryl or C₅ or C₆heterocycloalkyl;

is a single or double bond, wherein two double bonds are not adjacent;and

is a double bond or


2. The method of claim 1 wherein the compound is

or a pharmaceutically acceptable salt thereof.